Microassay diagnostic test method

ABSTRACT

The method of determining the total iron binding capacity (TIBC) of human blood involves removing the iron content of an aliquot of blood, adding a solution of radioactive ferric cations to the iron free aliquot, incubating the mixture at a pH in the range of 7 to 9 to effectuate coupling of ferric cations and protein, measuring the initial radioactivity of the mixture, contacting the mixture with anion exchange resin to produce a solution free of unassociated radioactive ferric cations and measuring residual radioactivity of said solution free of unassociated radioactive ferric cations.

United StatesPatent [191 Yang et a]. 1 Nov. 20, 1973 ['54] MICROASSAY DIAGNOSTIC TEST 3,666,854 5/1972 Eisentraut 23/230 B UX METHOD 3,667,915 6/1972 Klein 23/230 B Inventors: Shih-Tzy Yang, Justice; Sunny Edmund Egozi, Chicago; Sam S. Yanari, Park Forest, all of ill.

Assignee: Wilson Pharmaceutical & Chemical Corporation, Chicago, 111.

Filed: Nov. 17, 1911 Appl. No.: 199,737

References Cited UNITED STATES PATENTS 3/1968 Storey 23/230 11/1970 O'Malley et al 23/230 B Primary Examiner-Morris 0. Walk Assistant ExaminerR. E. Serwin Attorney-Max Dressler et al.

[57] ABSTRACT The method of determining the total iron binding capacity (TIBC) of human blood involves removing the iron content of an aliquot of blood, adding a solution of radioactive ferric cations to the iron free aliquot, incubating the mixture at a pH in the range of 7 to 9 to effectuate coupling of ferric cations and protein, measuring the initial radioactivity of the mixture, contacting the mixture with anion exchange resin to produce a solution free of unassociated radioactive ferric cations and measuring residual radioactivity of said solution free of unassociated radioactive ferric cations.

6 Claims, No Drawings 1 MICROASSAY DIAGNOSTIC TEST METHOD This invention relates to a method for determining the total iron binding capacity of the liquid portion of human blood. More particularly, it relates to a method of effecting a direct measurement of the total iron binding capacity of human blood which in cooperation with conventional unsaturated iron binding capacity (UlBC) measurements provides a highly accurate determination for clinical use in analysis of conditions of humans with myelophthisic, pernicious anemia and related diseases and is of particular urgency in pediatric medicine where only small volumes of serum samples can be obtained for analysis. Still more particularly, it relates to a method of removing iron naturally present in the liquid portion of humand blood as a component bound to proteins such as iron-binding Beta globulin to form iron free blood aliquots and then reforming iron bound blood proteins in the aliquots with radioactive iron so as to have an accurate measurement of the true total iron-binding capacity.

Briefly the method of the present invention comprises removing the iron content of an aliquot of said liquid portion of human blood, mixing with the resultant iron-free liquid portion of human blood an aqueous buffer solution containing citrate ions, bicarbonate ions and radioactive ferric ion to produce a mixture having a pH in the range of 7 to 9, incubating the mixture to effectuate coupling of radioactive ferric ion and transferrin, measuring the initial radioactivity of the mixture, contacting said mixture containing iron saturated transferrin and the excess iron present in the form of iron citrate complex with anion exchange resin to produce a solution containing iron essentially only in the form of iron saturated transferrin and measuring residual radioactivity of said contacted anion resin or said solution containing said iron saturated transferrin.

in analysis of conditions of human iron metabolism, three parameters, i.e., serum iron(SI), unsaturated iron binding capacity (UlBC) and total iron binding capacity (TIBC) must be known. Since TlBC is equal to UlBC plus SI, determinations generally are limited to two out of the three parameters.

Determination of iron binding capacity by known colorimetric methods, have involved cumbersome and tedious techniques. Most methods suffer from shortcomings, notably, lack of sensitivity and reproducibility and need for relatively large volume of serum samples. A typical colorimetric method makes use of the fact that the protein-iron complex exhibits a pink color and provides a basis for estimation photometrically by comparison with standards. The method gives an estimate of the unsaturated iron binding capacity (UlBC) by measuring the increase in color resulting from addition of iron salts. In view of the smallness of changes in optical density, the accuracy of these methods are not entirely satisfactory.

Methods involving radioactivity as the basis for analysis also are known for determination only of UlBC and SI but has not been developed for TlBC. UlBC determination by radioassay is generally reliable and suffi ciently sensitive. On the other hand, an independent determination by radioassay of S1, is not currently available. Sl, as determined by isotope dilution technique, such as is disclosed in the Herbert article in the Journal of Nuclear Medicine 82529-541, 1967, is inherently limited as to reliability and sensitivity.

A radioassay method, limited to measurement of the UlBC of blood serum, is shown in Storey et al. U.S. Pat. No. 3,372,992. The method consists of adding radioactive iron to a blood serum in excess of the amount required to react with the protein and then adding a sponge incorporating an anion exchange resin to react with the excess iron which has not reacted with the protein. The total radioactivity added to the serum is measured by a gamma ray well counter, then the serumradioactive iron solution is discarded and the washed sponge measured so as to be able to express the quantity of UlBC as the difference between the total weight of iron added to the serum and the percent sponge uptake. Such a method determines only the UlBC and does not provide sufiicient information for a complete diagnosis of a patients serum iro:n status.

It has now been discovered that by directly measuring the total iron binding capacity (TlBC) of an ironfree aliquot of the liquid portion of human blood, as

hereinafter set forth, and by independently measuring the UlBC of a separate aliquot of the liquid portion of human blood under conditions so that any variations will be of the same nature, means can be provided for an easy and more accurate procedure for radioassay and complete clinical diagnosis ofa patients serum iron function status.

More in detail, a method of this invention of determining the total iron binding capacity of the liquid portion of human blood containing transferrin protein molecules which comprises adjusting the acidity of an aliquot of said liquid portion of human blood to a pH in the range between 4 and 5.5, to effect dissolution of iron cations from the protein molecule, with a buffer solution containing citrate ion-iron isolating agent whose ferric-citrate complex has an affinity for anion resinycontacting said pl-l adjusted liquid with a strong base anion exchange resin, to remove substantially all and at least of the iron content of said aliquot, separating the iron-free liquid from said resin, mixing with said iron-free liquid an aqueous buffer solution containing citrate ions, bicarbonate ions and radioactive ferric ions, to produce a mixture having a pH in the range of 7 to 9, incubating the mixture to effectuate coupling of radioactive ferric ions and transferrin, measuring the initial radioactivity of the mixture, contacting said mixture containing iron saturated transferrin, and the excess iron present in the form of iron citrate complexes with anion exchange resin to produce a solution containing iron essentially only in the form of iron saturated transferrin, and measuring residual radioactivity of said contacted anion resin or said solution containing said iron saturated transferrin.

Use of citric acid in a buffer solution designed to adjust pH of the serum to a pH in the range of 4-5.5 for effecting dissociation of iron from the protein content of a blood serum or blood plasma is an element essential for substantially complete removal of iron. Other polycarboxylic acids, such as succinic acid, aconitic acid, etc, over a period of up to 180 minutes only effect isolation of the iron in amounts of less than 50%. Citric acid isolates the dissociated ferric ions in a complex which in a period of i0 minutes at pH 4 permits removal of about 88.5% and at pl-ll 5 permits removal of about 94.3% of the ferric ions. At pH 6, the iron removal in 10 minutes is only about 54% and even over a period of minutes the maximum iron removal is about 60%. In order to accomplish substantially complete iron removal, blood serum can be treated at pH of 4 and 5.5 over a period of 10 minutes to 120 minutes with the result that the iron removal will reach amounts of the order of 98%. A composition useful in dissociat ing and removing the iron from human blood serum comprises a buffer solution-suspension containing 0.02M to 0.06l7M sodium citrate, 0.1M to 0.3M sodium chloride, and 0.3 ml. of 20 to 50 mesh anion exchange resin per ml. of buffer solution.

While the treatment may be extended over a period up to 180 minutes to insure substantially complete iron removal, it is preferred to limit the treatment to a pH between 5 and 5.5 and to a period of IO to 30 minutes.

After treatment of the blood serum with citric acidcontaining buffer solution, the iron present in the form of iron-citrate complex is removed by contact of the solution with an anion exchange resin of strong base strength properties such as those sold by Rohm and Haas under the tradename Amberlite IRA-410, etc., a type of resin such as is described in US. Pat. No. 2,591,573.

In the step of reforming the iron-transferrin product, use of sodium bicarbonate in an alkaline buffer solution designed to adjust the alkalinity of iron-free serumradioactive iron solution mixture to a pH in the range of 7 to 9, is an element essential to formation of stable transferrin iron complex. ln the presence of bicarbonate ion, even in the presence of citrate ion for forming iron citrate complex of the iron in amounts in excess of that required to saturate the transferrin, the quantity of iron which will bind to transferrin is almost identical to theoretical quantities whereas, for example, known barbital buffers, show drastic variations from theoretical quantities.

A preferred type of composition useful in binding, i.e., reassociating iron with the protein content of ironfree blood serum comprises a solution of pH 8.6, containing 0.1M to 0.3M tris (hydroxymethyl) aminomethane and 0.2M to 0.5M sodium bicarbonate.

Effectiveness of dissociation of iron from a blood serum and the reproducibility of analysis may be established by depositing in a series of test tubes to obtain average figures, human blood serum previously treated with radioactive iron containing solution to induce ad dition of tracer amounts thru reaction with the unsaturated iron binding capacity of the serum. When 0.1 ml. of human serum containing 11 nanograms of radioactive iron, and a dissociation buffer solution consisting of 0.4 ml. ofa 0.02M citrate and 0.1M Na Cl (pH-4.5) are mixed, a resultant solution is obtained with a pH of approximately 5. After an incubation period additional buffer solution containing 0.02M citric acid and 0.1M Na Cl and 0.6 ml. volume of IRA-4 l0 particulate anion exchange resin, is added. After agitation for from to 180 minutes, the solution may be decanted and the resin washed twice with 5 ml. of iron-free water prior to measurement of the radioactivity of the resin with associated iron citrate.

The original human blood serum, having a radioactivity measurement of 16963 CPM per 0.5 mL, after various iron dissociation reaction periods and contact with resin, shows a resin uptake by the anion exchange resin as follows:

TABLE 1 Dissociation at pH 5.0

Time

Resin Uptake %Uptake Mins. CPM. O O 0 5 16.009 94.4 l5 l6,363 96.5 30 l6,645 98.1 60 16,615 98.0 16,74l 98.7 I20 l6,783 98.9

The data indicates substantially complete removal of iron from the blood serum.

Effectiveness of reassociation of iron dissociated by the method indicated previously, but not eliminated therefrom, may be illustrated by treatment of incubated solutions corresponding to those whose dissociation is shown in table l, by adding to such a mixture 1 ml. of a buffer solution of pH 8.6 containing 0.05M tris (hydroxymethyl) aminomethane-0.1M sodium bicarbonate. Addition of this buffer solution results in producing a mixture having a pH of approximately 8.2. After various incubation periods up to 60 minutes, 0.6 ml. volume of particulate anion exchange resin is added to each mixture. After an agitation period of about 10 minutes for each of the resultant slurry, the solution is decanted, the resin washed twice with 5 ml. of iron'free water and the washed resin checked to determine the radioactivity of the washed resin.

The resin uptake is shown in the following table.

TABLE 2 RE-ASSOClATlON AT pH 8.2

Time Resin Uptake %Uptake Mins. CPM.

0 16,615 98.0 5 I05 0.6 [5 I I2 0.7 30 92 0.5 60 0.8

More in detail, a preferred embodiment of the method of this invention consists of the steps of treating an aliquot of blood to clot the blood, and then separating the serum. An aliquot of the blood serum is mixed with a citric acid containing buffer solution to adjust.

the acidity thereof to a pH in the range between 4 and 5.5. The pH adjusted serum is passed thru a tower containing particulate anion exchange resin to remove the iron-citrate complex and produce a iron-free eluate containing blood protein known as transferrin. The aliquot of iron-free eluate is mixed with an aqueous solution containing radioactive ferric ion in a bicarbonate buffer solution in quantities to produce a mixture having an acidity in the pH range of 8 to 9.5 and a total radioactivity of 0.05 to 0.15 microcurie per milliliter of mixture. The mixture is incubated for a period of about '10 minutes at a temperature in the range between about 60F and about 90F, to effect complete saturation of the transferrin content of the mixture. The radioactivity of the incubated mixture is measured in a conventional gamma ray counter. The incubated mixture is then passed thru a column containing particulate anion exchange resin to remove the excess radioactive iron present in the form of iron citrate complex. The eluate containing radioactive iron-transferrin is deposited ina tube and inserted into the well of a conventional gamma ray counter for measuring the radioactivity whereby the quantity of ferric iron bound to the transferrin, i.e., the TlBC is determinable. The radioactivity of the resin bound complex may be similarly determined.

When the unsaturated iron binding capacity (UIBC) is to be determined for analysis purposes, a 0.1 ml. aliquot of blood serum is mixed with 0.4 ml. of buffer solution of pH 8.6 containing 0.02M sodium citrate, 0.lM sodium chloride, 0. l M tris (hydroxymethyl) aminomethane 0.2M sodium bicarbonate, 1 ml. of radioactive iron solution ("Fe) containing 24 mg./liter of iron in the form of ferric ammonium sulfate, 0.02M sodium citrate, and 0.1M sodium chloride.

After agitation for a period of, for example, minutes, 0.6 ml. volume of particulate anion exchange.

resin (Amberlite lRA-4l0) is added and agitation continued for another 15 minutes.

The suspension is then allowed to settle and supernatant liquid separated from the resin which has tied up iron not bound to serum protein.

Supernatant is then checked for radioactivity in a gamma ray well counter to determine the radioactivity to be compared to the radioactivity of the solution added to the serum.

The following example is presented to illustrate the practice of the method of determining the total iron binding capacity of serum protein in greater detail and is not to be construed as presenting limitations on the invention.

Glass test tubes of a 13 X l00 mm size are acid washed for assured cleanliness.

A 2 ml. aliquot of acidic buffer solution consisting of 0.02M sodium citrate and 0.1M sodium chloride and having a pH of 4.5 and containing 0.6 ml. volume of particulate strong base anion exchange resin (Amberlite IRA-410) of to 50 mesh size is pipetted into the test tube.

A 0.5 ml. aliquot of blood serum is introduced into the tube, the tube is closed and then agitated by a shaker for example, 30 minutes.

After standing for a period, following agitation, during which the solidstresin) settles, a 0.5 ml. aliquot of supernatant blood serum solution is withdrawn and introduced into a second test tube. The solution in the second test tube is mixed with 1 ml. of an alkaline buffer solution prepared from 0.5 ml. of radioactive ferric chloride solution (Fe C1 diluted with 2.5 ml. of solution of ferric ammonium sulfate containing 0.02M sodium citrate and 0.1M sodium chloride and further diluted to 100 ml. total volume with abicarbonate buffer solution containing 0.1M tris (hydroxymethyl) aminomethane 0.2M sodium bicarbonate adjusted to pH 8.6 with sodium hydroxide, to reduce the radioactivity to the 0.1 microcurie or less level to conform to the general licensing regulations of the Atomic Energy Commission.

The mixture is incubated at room temperature for about 15 minutes and 0.6 ml. volume of 20 to 50 mesh size Amberlite IRA-410 anion exchange resin wetted with deionized water, is added and the agitation continued for another 15 minutes.

The mixture is centrifuged to separate clear supernatant solution. Radioactivity of the clear supernatant solution can be determined in a gamma well counter. The radioactivity of the mixture containing the total added -radioactive iron was 21769 CPM. This represents 607 nanogram of ferric iron. The CPM of the clear supernatant solution was 10528 CPM indicating that the TIBC is 294 nanograms per 0.1 ml. of

serum.

Although preferred embodiments of the present invention have been described, it will be understood that the description is designed to be illustrative rather than restrictive, as details may be modified or changed without departing from the spirit or the scope of the invention.

We claim:

1. A method of determining the total iron binding capacity of the liquid portion of human blood containing transferrin protein molecules which comprises removing the iron content of an aliquot of said liquid portion of human blood, mixing with the resultant iron-free liquid portion of human blood an aqueous buffer solution containing citrate ions, bicarbonate ions and radioactive ferric ion to produce a mixture having a pH in the range of 7 to 9, incubating the mixture to effectuate coupling of radioactive ferric ion and transferrin, measuring the initial radioactivity of the mixture, contacting said mixture containing iron saturated transferrin and the excess iron present in the form of iron citrate complex with anion exchange resin to produce a solution containing iron essential only in the form of iron saturated transferrin and measuring residual radioactivity of said contacted anion resin or said solution containing said iron saturated transferrin.

2. A method according to claim 1 wherein said liquid portion of human blood is serum.

3. A method according to claim 1 wherein said liquid portion of human blood is plasma.

4. A method according to claim 1 wherein said acidic iron isolating agent is citric acid.

5. A method of determining the natural iron content of human blood protein which comprises subtracting from the TlBC content determined in accordance with claim 1 the UIBC determined by a conventional method.

6. A method of determining the total iron binding capacity of the liquid portion of human blood containing transferrin protein molecules which comprises adjusting the acidity of an aliquot of said liquid portion of human blood to a pH in the range between 4 and 5.5 with a buffer solution containing citrate ion-iron isolating agent whose ferriccitrate complex has an affinity for anion resin, contacting said pH adjusted liquid with a strong base anion exchange resin, separating the ironfree liquid from said resin, mixing with said iron-free liquid an aqueous buffer solution containing citrate ions, bicarbonate ions and radioactive ferric ions, to produce a mixture having a pH in the range of 7 to 9, incubating the mixture to effectuate coupling of radioactive ferric ions and transferrin, measuring the initial radioactivity of the mixture, contacting said mixture containing iron saturated transferrin and the excess iron present in the form of iron ferric citrate complex with anion exchange resin to produce a solution containing iron essentially only in the form of iron saturated transferrin, and measuring residual radioactivity of said contacted anion resin or said solution containing said iron saturated transferrin.

l l l I 

2. A method according to claim 1 wherein said liquid portion of human blood is serum.
 3. A method according to claim 1 wherein said liquid portion of human blood is plasma.
 4. A method according to claim 1 wherein said acidic iron isolating agent is citric acid.
 5. A method of determining the natural iron content of human blood protein which comprises subtracting from the TIBC content determined in accordance with claim 1 the UIBC determined by a conventional method.
 6. A method of determining the total iron binding capacity of the liquid portion of human blood containing transferrin protein molecules which comprises adjusting the acidity of an aliquot of said liquid portion of human blood to a pH in the range between 4 and 5.5 with a buffer solution containing citrate ion-iron isolating agent whose ferriccitrate complex has an affinity for anion resin, contacting said pH adjusted liquid with a strong base anion exchange resin, separating the iron-free liquid from said resin, mixing with said iron-free liquid an aqueous buffer solution containing citrate ions, bicarbonate ions and radioactive ferric ions, to produce a mixture having a pH in the range of 7 to 9, incubating the mixture to effectuate coupling of radioactive ferric ions and transferrin, measuring the initial radioactivity of the mixture, contacting said mixture containing iron saturated transferrin and the excess iron present in the form of iron ferric citrate complex with anion exchange resin to produce a solution containing iron essentially only in the form of iron saturated transferrin, and measuring residual radioactivity of said contacted anion resin or said solution containing said iron saturated transferrin. 